Specific Regulation Of Glycine Receptor α1^ins Subunit By MGluR5/ERK Signaling In Spinal Cord Dorsal Horn

Objective:Inhibitory glycinergic neurotransmission in adult spinal cord is primarily mediated by glycine receptors（GlyRs）containingα1 subunit.Alternative splicing can generateα1^ins,a longerα1 variant in which 8 amino acids are inserted into the intracellular large loop between transmembrane TM3 and TM4 domains.However,the functional significance and regulation mechanisms of this splice variant remain to be elucidated.This study aimed to reveal the role ofα1ⁱnsns subunit in spinal nociceptive transduction and its pathological significance.Methods:Co-immunoprecipitation and GST pull-down experiments were conducted to discover the molecular chaperones ofα1ⁱnsns subunit.Immunohistochemistry experiments were performed to examineα1^ins expression in the dorsal horn of spinal cord.The intracellular trafficking ofα1^ins was investigated by immunocytochemistry experiments in cultured spinal neurons.GlyRs-mediated inhibitory postsynaptic currents（GlyRs-IPSCs）were recorded by whole-cell patch clamp techniques in lamina II neurons within the spinal cord slices of mice.LC-MS/MS method was used to identify the phosphorylation and ubiquitination sites onα1^ins.Inflammatory pain was induced by injection of formalin into hindpaws.The pain thresholds were measured to reveal the role ofα1ⁱnsns in inflammatory pain.Results:（1）α1^ins was abundantly expressed in the superficial dorsal horn of spinal cord,located at inhibitory postsynaptic membrane and involved in the generation of glycinergic synaptic currents.The downregulation ofα1^ins expression decreased the amplitudes of GlyRs-mediated miniature IPSCs（mIPSCs）,without influence on GlyRs-mIPSCs frequencies;（2）α1^ins played an important role in the modification and integration of nociceptive signals.The shRNA designed against mouseα1^ins(shRNA-α1^ins)significantly increased the neuronal excitability and resulted in the nociceptive sensitization to mechanical,heat and cold stimuli;（3）α1^ins subunit was a specific substrate of mGluR5.Activation of mGluR5 selectively decreased the amplitudes ofα1^ins currents,with no detectable changes ofα1 andα3 currents;（4）Extracellular signal-regulated kinase（ERK）served as the key downstream signaling component for mGluR5 to inhibitα1^ins.By activating ERK,mGluR5 suppressed the glycinergic synaptic transmission.shRNA-α1^ins blocked mGluR5from reducing GlyRs-IPSCs,while shRNA against GlyRsα3 subunit had no effect on mGluR5-mediated inhibition of GlyRs-IPSCs;（5）The active ERK specifically interacted withα1^ins,but didn’t bind to GlyRsα1,α3 orβsubunits;（6）The amino acid sequence between 316-334 residues ofα1^ins was responsible for ERK binding.A TAT-fused synthetic peptide(TAT-pep-α1^ins)that mimicked theα1^ins binding sequence disturbedα1^ins/ERK interaction in vitro and in vivo;（7）LC-MS/MS analysis showed that ERK directly phosphorylatedα1ⁱnsns at Ser380;（8）Ser380phosphorylation favored the ubiquitination modification ofα1^ins;（9）Lys379 was the major site for ubiquitin modification onα1^ins;（10）Lys379 ubiquitination promoted the interaction ofα1^ins with epidermal growth factor receptor substrate 15（Eps15）,which induced the endocytosis ofα1ⁱnsns from plasma membrane;（11）Through mGluR5/ERK pathway,peripheral inflammation enhanced the binding of ERK toα1^ins,which increased Ser380 phosphorylation,decreased the membrane expression ofα1^ins,reduced the amplitudes of GlyRs-IPSCs and led to glycinergic disinhibition;（12）Intrathecal application of TAT-pep-α1^ins in inflamed animals blocked ERK phosphorylation of Ser380 and specifically enhanced the amplitudes of GlyRs-IPSCs.The synaptic responses mediated by glutamatergic receptors and GABA_A receptors were insensitive to TAT-pep-α1^ins;（13）Intrathecal injection of TAT-pep-α1^ins alleviated spontaneous pain induced by mGluR5 and the second-phase painful responses induced by Formalin in a dose-dependent manner.Conclusion:mGluR5/ERK signaling pathway specifically downregulatedα1^ins subunit-mediated inhibitory glycinergic synaptic transmission in spinal cord dorsal horn.Interference with the molecular interaction between ERK andα1^ins produced an analgesic action.